Glucoamylase variants

ABSTRACT

The invention relates to a variant of a parent fungal glucoamylase, which exhibits improved thermal stability and/or increased specific activity using saccharide substrates.

FIELD OF THE INVENTION

The present invention relates to glucoamylase variants (mutants) ofparent AMG, in particular with altered thermal stability and/or alteredspecific activity suitable for, e.g., starch conversion, e.g., forproducing glucose from starch. More specifically, the present inventionrelates to glucoamylase enzyme variants and the use of such variantenzymes.

BACKGROUND OF THE INVENTION

Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is anenzyme, which catalyzes the release of D-glucose from the non-reducingends of starch or related oligo- and polysaccharide molecules.Glucoamylases are produced by several filamentous fungi and yeasts, withthose from Aspergillus being commercially most important.

Commercially, the glucoamylase enzyme is used to convert corn starchwhich is already partially hydrolyzed by an alpha-amylase to glucose.The glucose is further converted by glucose isomerase to a mixturecomposed almost equally of glucose and fructose. This mixture, or themixture further enriched with fructose, is the commonly used highfructose corn syrup commercialized throughout the world. This syrup isthe world's largest tonnage product produced by an enzymatic process.The three enzymes involved in the conversion of starch to fructose areamong the most important industrial enzymes produced.

One of the main problems that exist with regard to the commercial use ofglucoamylase in the production of high fructose corn syrup is therelatively low thermal stability of glucoamylase. Glucoamylase is not asthermally stable as alpha-amylase or glucose isomerase and it is mostactive and stable at lower pH's than either alpha-amylase or glucoseisomerase. Accordingly, it must be used in a separate vessel at a lowertemperature and pH.

Glucoamylase from Aspergillus niger has a catalytic (aa 1-440) and astarch binding domain (aa 509-616) separated by a long and highlyO-glycosylated linker (Svensson et al. (1983), Carsberg Res. Commun. 48,529-544, 1983 and (1986), Eur. J. Biochem. 154, 497-502). The catalyticdomain (aa 1471) of glucoamylase from A. awamori var. X100 adopt an(alpha/alpha)₆-fold in which six conserved alpha→alpha loop segmentsconnect the outer and inner barrels (Aleshin et al. (1992), J. Biol.Chem. 267, 19291-19298). Crystal structures of glucoamylase in complexwith 1-deoxynojirimycin (Harris et al. (1993), Biochemistry, 32,1618-1626) and the pseudotetrasaccharide inhibitors acarbose andD-glucodihydroacarbose (Aleshin et al. (1996), Biochemistry 35,8319-8328) furthermore are compatible with glutamic 0.35 acids 179 and400 acting as general acid and base, respectively. The crucial role ofthese residues during catalysis have also been studied using proteinengineering (Sierks et al. (1990), Protein Engng. 3, 193-198; Frandsenet al. (1994), Biochemistry, 33, 13808-13816). Glucoamylase-carbohydrateinteractions at four glycosyl residue binding subsites, −1, +1, +2, and+3 are highlighted in glucoamylase-complex structures (Aleshin et al.(1996), Biochemistry 35, 8319-8328) and residues important for bindingand catalysis have been extensively investigated using site-directedmutants coupled with kinetic analysis (Sierks et al. (1989), ProteinEngng. 2, 621-625; Sierks et al. (1990), Protein Engng. 3, 193-198;Berland et al. (1995), Biochemistry, 34, 10153-10161; Frandsen et al.(1995), Biochemistry, 34, 10162-10169.

Different substitutions in A. niger glucoamylase to enhance the thermalstability have been described: i) substitution of alpha-helicalglycines: G137A and G139A (Chen et al. (1996), Prot. Engng. 9, 499-505);ii) elimination of the fragile Asp-X peptide bonds, D257E and D293E/Q(Chen et al. (1995), Prot. Engng. 8, 575-582); prevention of deamidationin N182 (Chen et al. (1994), Biochem. J. 301, 275-281); iv) engineeringof additional disulphide bond, A246C (Fierobe et al. (1996),Biochemistry, 35, 8698-8704; and v) introduction of Pro residues inposition A435 and S436 (Li et al. (1997), Protein Engng. 10, 1199-1204.Furthermore Clark Ford presented a paper on Oct. 17, 1997, ENZYMEENGINEERING 14, Beijing/China October 12-17, 97, Abstract number:Abstract book p. 0-61. The abstract suggests mutations in positionsG137A, N20C/A27C, and S30P in an (not disclosed) Aspergillus awamoriglucoamylase to improve the thermal stability.

BRIEF DISCLOSURE OF THE INVENTION

The object of the present invention is to provide improved glucoamylasevariants with altered properties, especially with alteredthermostablility and/or altered specific activity suitable for use in,e.g., the saccharification step in starch conversion processes.

The term “a glucoamylase variant with altered thermostability” means inthe context of the present invention a glucoamylase variant, which has ahigher or lower T_(1/2) (half-time) than the corresponding parentglucoamylase. The determination of T½ (Method I and Method II) isdescribed in the “Materials & Methods” section of WO 00/04136.

The term “a glucoamylase variant with altered specific activity” meansin the context of the present invention a glucoamylase variant withaltered specific activity towards the alpha-1,4 linkages in thesaccharide in question. The specific activity is determined as k_(cat)or AGU/mg (measured as described in the “Materials & Methods” section ofWO 00/04136). An increased specific activity means that the k_(cat) orAGU/mg values are higher when compared to the k_(cat) or AGU/mg values,respectively, of the corresponding parent glucoamylase.

The inventors of the present invention have provided a number ofimproved variants of a parent glucoamylase with altered thermostabilityand/or altered specific activity in comparison to the parentcorresponding enzyme. The altered thermal stability is obtained bysubstituting selected positions in a parent glucoamylase. This will bedescribed in details below.

Nomenclature

In the present description and claims, the conventional one-letter andthree-letter codes for amino acid residues are used. For ease ofreference, glucoamylase variants of the invention are described by useof the following nomenclature:

Original amino acid(s):position(s):substituted amino acid(s)

According to this nomenclature, for instance the substitution of alaninefor asparagine in position 30 is shown as:

-   -   Ala30Asn or A30N        a deletion of alanine in the same position is shown as:    -   Ala30* or A30*        and insertion of an additional amino acid residue, such as        lysine, is shown as:    -   Ala30AlaLys or A30AK

A deletion of a consecutive stretch of amino acid residues, such asamino acid residues 30-33, is indicated as (30-33)* or Δ(A30-N33).

Where a specific glucoamylase contains a “deletion” in comparison withother glucoamylases and an insertion is made in such a position this isindicated as:

-   -   *36Asp or *36D        for insertion of an aspartic acid in the “deletion” position 36.        Multiple mutations are separated by plus signs, i.e.:    -   Ala30Asp+Glu34Ser or A30N+E34S        representing mutations in positions 30 and 34 substituting        alanine and glutamic acid for asparagine and serine,        respectively. Multiple mutation may also be separated as        follows, i.e., meaning the same as the plus sign:    -   Ala30Asp/Glu34Ser or A30N/E34S

When one or more alternative amino acid residues may be inserted in agiven position it is indicated as

-   -   A30N,E or A30N/E, or A30N or A30E

Furthermore, when a position suitable for modification is identifiedherein without any specific modification being suggested, it is to beunderstood that any amino acid residue may be substituted for the aminoacid residue present in the position. Thus, for instance, when amodification of an alanine in position 30 is mentioned, but notspecified, it is to be understood that the alanine may be deleted orsubstituted for any other amino acid, the latter substitution may alsobe indicated as:

-   -   A30R,N,D,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V or A30X, where X        denotes any other amino acid.

DETAILED DISCLOSURE OF THE INVENTION

A goal of the work underlying the present invention was to alter thethermal stability and/or alter the specific activity of particularglucoamylases which are obtainable from fungal organisms, in particularstrains of the Aspergillus genus and which themselves had been selectedon the basis of their suitable properties in starch conversion oralcohol fermentation.

Identifying Positions and/or Regions to be Mutated to Obtain AlteredThermostability and/or Altered Specific Activity

Molecular dynamics (MD) simulations indicate the mobility of the aminoacids in the protein structure (see McCammon, J A and Harvey, S C.,(1987), “Dynamics of proteins and nucleic acids”, Cambridge UniversityPress). Such protein dynamics are often compared to the crystallographicB-factors (see Stout, G H and Jensen, L H, (1989), “X-ray structuredetermination”, Wiley). By running the MD simulation at differentprotonation states of the titrate able residues, the pH related mobilityof residues are simulated. Regions having the highest mobility orflexibility (here isotropic fluctuations) are selected for randommutagenesis. It is here understood that the high mobility found incertain areas of the protein, can be thermally improved by substitutingresidues in these residues. The substitutions are directed againstresidues that will change the dynamic behaviour of the residues to e.g.bigger side-chains and/or residues, which have capability of formingimproved contacts to residues in the near environment. The AMG fromAspergillus niger was used for the MD simulation. How to carry out MDsimulation is described in the Materials & Methods” section below.)Regions found to be of interest for increasing the specific activityand/or improved thermostability are the regions in proximity to theactive site. Regions positioned in between the α-helixes, and which mayinclude positions on each side of the N- and C-terminal of theα-helixes, at the substrate-binding site is of importance for theactivity of the enzyme.

Rhizopus, Talaromyces, such as Talaromyces emersoni (dislosed in WO99/28448), and Thielavia have high specific activity towardsmaltodextrins, including maltose and maltohepatose. Therefore, regionsbeing of special interest are those involved in transferring specificactivity.

The present inventors find that it is in fact possible to alter thethermal stability and/or to alter the specific activity of a parentglucoamylase by modification of one or more amino acid residues of theamino acid sequence of the parent glucoamylase. The present invention isbased on this finding.

Accordingly, in a first aspect the present invention relates to animproved variant of a parent glucoamylase comprising one or moremutations in the regions and positions described further below.

Parent Glucoamylases

Parent glucoamylase contemplated according to the present inventioninclude fungal glucoamylases, in particular fungal glucoamylasesobtainable from an Aspergillus strain, such as an Aspergillus niger orAspergillus awamori glucoamylases and variants or mutants thereof,homologous glucoamylases, and further glucoamylases being structurallyand/or functionally similar to the amino acid sequence shown in SEQ IDNO: 2 of WO 00/04136 (Novo Nordisk). Specifically contemplated are theAspergillus niger glucoamylases G1 and G2 disclosed in Boel et al.(1984), “Glucoamylases G1 and G2 from Aspergillus niger are synthesizedfrom two different but closely related mRNAs”, EMBO J. 3 (5), p.1097-1102. The G2 glucoamylase is disclosed as SEQ ID NO: 2 in WO00/04136 (Novo Nordisk). The G1 glucoamylase is disclosed as SEQ ID NO:13 of WO 00/04136. Another AMG backbone contemplated is Talaromycesemersonii, especially Talaromyces emersonii DSM disclosed in WO 99/28448(Novo Nordisk).

Commercially Available Parent Glucoamylases

Commercially available parent glucoamylases include AMG from Novozymes,and also glucoamylase from the companies Genencor Int., Inc., USA, andGist-Brocades (DSM), Delft, The Netherlands.

Glucoamylase Variants

In the first aspect the invention relates to a variant of a parentglucoamylase comprising one or more mutation(s) in position(s) orregion(s) corresponding to the following position(s) or region(s) in theamino acid sequence published as SEQ ID NO: 2 in WO 00/04136:

-   a) said variant comprising one or more insertion(s) in: Region:    1-35, Region: 40-58, Region: 60-62, Region: 73-80, Position: 93,    Region: 95-101, Region: 103-121, Region: 123-124, Region: 126-127,    Region: 170-175, Region: 177-184, Region: 200-212, Region: 234-246,    Region: 287-312, Region: 314-319, Region: 334-339, Position: 341,    Region: 354-356, Position: 358, Region: 360-374, Region: 388-392,    Position: 94, Region: 396-401, Region: 403-407, Region: 409-414,    Region: 445-449, Region: 452-467, Region: 469-470, and/or in a    corresponding position or region in a homologous glucoamylase which    displays at least 60% homology with the amino acid sequences shown    in SEQ ID NO: 2 of WO 00/04136, or-   b) said variant comprising one or more substitution(s), insertion(s)    and/or deletion(s) in: Region: 36-39, Region: 63-65, Position 67,    Region: 69-71, Region: 81-92, Region: 128-169, Region: 85-188,    Region: 190-199, Region: 213-222, Region: 224-226, Region: 228-233,    Region: 247-271, Region: 273-286, Region: 320-333, Region: 343-344,    Region: 346-347, Region: 349-351, Region: 375-378, Region: 380-385,    Position: 387, Position: 415, Region: 417-424, Position: 426,    Region: 428-443, Region: 471-485, Region: 487489, Region: 491-493,    Region: 495-616, and/or in a corresponding position or region in a    homologous glucoamylase which displays at least 60% homology with    the amino acid sequence shown in SEQ ID NO: 2 of WO 00/04136, except    the following amino acid substitutions: A39V, P128S, G137A, G139A,    D153N, S185H, G251A, D257E, E259D, E259Q, C320A, D375C, G383A,    W417F, S431C, A435P, S436P, W437F, A442T, A471C, A479C, T480C,    P481C, A495T and A495P.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 1-35. Specific preferred positions contemplated include oneor more of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 40-58. Specific preferred positions contemplated include oneor more of: 40, 41, 42, 43, 44, 45, 46, 47, 49, 49, 50, 51, 52, 53, 54,55, 56, 57, 58.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 60-62. Specific preferred positions contemplated include oneor more of: 60, 61, 62.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 73-80.

Specific preferred positions contemplated include one or more of: 73,74, 75, 76, 77, 78, 79, 80.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 95-101. Specific preferred positions contemplated includeone or more of: 95, 96, 97, 98, 99, 100, 101.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 103-121. Specific preferred positions contemplated includeone or more of: 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 123-124. Specific preferred positions contemplated includeone or more of: 123, 124.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 126-127. Specific preferred positions contemplated includeone or more of: 126, 127.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 170-175. Specific preferred positions contemplated includeone or more of: 170, 171, 172, 173, 174, 175.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 177-184. Specific preferred positions contemplated includeone or more of: 177, 178, 179, 180, 181, 182, 183, 184.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 200-212. Specific preferred positions contemplated includeone or more of: 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 234-246. Specific preferred positions contemplated includeone or more of: 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 287-312. Specific preferred positions contemplated includeone or more of: 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,312.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 314-319. Specific preferred positions contemplated includeone or more of: 314, 315, 316, 317, 318, 319.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 334-339. Specific preferred positions contemplated includeone or more of: 334, 335, 336, 337, 338, 339. Another specific preferredposition is 341.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 354-356. Specific preferred positions contemplated includeone or more of: 354, 355, 356. Another specific preferred position is358.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 360-374. Specific preferred positions contemplated includeone or more of: 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 388-392. Specific preferred positions contemplated includeone or more of: 388, 389, 390, 391, 392. Another specific preferredposition is 394.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 396-401. Specific preferred positions contemplated includeone or more of: 396, 397, 398, 399, 400, 401.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 403-407. Specific preferred positions contemplated includeone or more of: 403, 404, 405, 406, 407.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 409-414. Specific preferred positions contemplated includeone or more of: 409, 410, 411, 412, 413, 414.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 445-449. Specific preferred positions contemplated includeone or more of: 445, 446, 447, 448, 449.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 452-467. Specific preferred positions contemplated includeone or more of: 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462,463, 464, 465, 466, 467.

In an embodiment, the region where one or more insertion(s) is made inthe Region: 469-470. Specific preferred positions contemplated includeone or more of: 469, 470.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made is the Region: 36-39. Specificpreferred positions contemplated include one or more of: 36, 37, 38, 39,except the amino acid substitution: A39V.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made is the Region: 63-65. Specificpreferred positions contemplated include one or more of: 63, 64, 65.Another specific preferred position is 67.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made is the Region: 69-71. Specificpreferred positions contemplated include one or more of: 69, 70, 71.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made is the Region: 81-92. Specificpreferred positions contemplated include one or more of: 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made is the Region: 128-169. Specificpreferred positions contemplated include one or more of: 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, except the aminoacid substitution(s) P128S, G137A, G139A, and D153N.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 185-188. Specificpreferred positions contemplated include one or more of: 185, 186, 187,188, except the amino acid substitution(s) S185H.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 190-199. Specificpreferred positions contemplated include one or more of: 190, 191, 192,193, 194, 195, 196, 197, 198, 199.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 213-222. Specificpreferred positions contemplated include one or more of: 213, 214, 215,216, 217, 218, 219, 220, 221, 222.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 224-226. Specificpreferred positions contemplated include one or more of: 224, 225, 226.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 228-233. Specificpreferred positions contemplated include one or more of: 228, 229, 230,231, 232, 233.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 247-271. Specificpreferred positions contemplated include one or more of: 247, 248, 249,250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263,264, 265, 267, 268, 269, 270, 271, except the amino acid substitution(s)G251A, D257E, E259D, and E259Q.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 273-286. Specificpreferred positions contemplated include one or more of: 273, 274, 275,276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 320-333. Specificpreferred positions contemplated include one or more of: 320, 321, 322,323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, except the aminoacid substitution(s) C320A.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 343-244. Specificpreferred positions contemplated include one or more of: 343, 344.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 346-347. Specificpreferred positions contemplated include one or more of: 346, 347.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 349-351. Specificpreferred positions contemplated include one or more of: 349, 350, 351.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 375-378. Specificpreferred positions contemplated include one or more of: 375, 376, 377,378, except the amino acid substitution(s) D375C.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 380-385. Specificpreferred positions contemplated include one or more of: 380, 381, 382,383, 384, 385, except the amino acid substitution(s) G383A. Otherspecific preferred positions contemplated include: 387, 415.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 417-424. Specificpreferred positions contemplated include one or more of: 417, 418, 419,420, 421, 422, 423, 424, except the amino acid substitution(s) W417F.Another specific preferred position contemplated includes: 426.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 428-443. Specificpreferred positions contemplated include one or more of: 428, 429, 430,431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, exceptthe amino acid substitution(s) S431C, A435P, S436P, W437F, A442T.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 471-485. Specificpreferred positions contemplated include one or more of: 471, 472, 473,474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, except theamino acid substitution(s) A471C, A479C, T480C, P481C.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 487489. Specificpreferred positions contemplated include one or more of: 487, 488, 489.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 491493. Specificpreferred positions contemplated include one or more of: 491, 492, 493.

In an embodiment, the region where one or more substitution(s),insertion(s) and/or deletion(s) is made in the Region: 495-616. Specificpreferred positions contemplated include one or more of: 495, 496, 497,498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511,512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525,526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539,540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553,554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567,568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581,582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595,596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609,610, 611, 612, 613, 614, 615, 616, except the amino acid substitution(s)A495T and A495P.

In a second aspect of the invention relates to a DNA constructcomprising a DNA sequence encoding a glucoamylase variant of the firstaspect. A third aspect of the invention relates to a recombinantexpression vector which carries a DNA construct according to the secondaspect and as defined elsewhere herein.

A fourth aspect relates to a cell which is transformed with a DNAconstruct according to the second aspect or a vector according to thethird aspect.

In a fifth aspect the invention relates to a cell according to thefourth aspect, which is a microorganism, such as a bacterium or afungus. A preferred embodiment relates to a cell of this aspect, whichis a protease deficient Aspergillus oryzae or Aspergillus niger.

A sixth aspect relates to a process for converting starch or partiallyhydrolyzed starch into a syrup containing dextrose, said processincluding the step saccharifying starch hydrolyzate in the presence of aglucoamylase variant according to the first aspect. Preferably thedosage of glucoamylase is present in the range from 0.05 to 0.5 AGU pergram of dry solids. In a preferred embodiment of this aspect the processcomprises saccharification of a starch hydrolyzate of at least 30percent by weight of dry solids. In another preferred embodiment, thesaccharification is conducted in the presence of a debranching enzymeselected from the group of pullulanase and isoamylase, preferably apullulanase derived from Bacillus acidopullulyticus or Bacillusderamificans or an isoamylase derived from Pseudomonas amyloderamosa.Still in another embodiment, the saccharification is conducted at a pHof 3 to 5.5 and at a temperature of 60-80° C., preferably 63-75° C., for24 to 72 hours, preferably for 36-48 hours at a pH from 4 to 4.5.

A seventh aspect relates to a method of saccharifying a liquefied starchsolution, which method comprises:

-   -   (i) a saccharification wherein one or more enzymatic        saccharification stages takes place, and    -   (ii) one or more high temperature membrane separation steps,        wherein the enzymatic saccharification is carried out using a        glucoamylase variant according to the first aspect.

Several aspects of the invention relate to uses of a glucoamylasevariant according to the first aspect, such as the use in a starchconversion process; the use in a continuous starch conversion process,preferably wherein the continuous starch conversion process include acontinuous saccharification process according to the seventh aspect; theuse in a process for producing oligosaccharides; the use in a processfor producing specialty syrups; the use in a process for producingethanol for fuel; the use in a process for producing a beverage; andfinally the use in a fermentation process for producing organiccompounds, such as citric acid, ascorbic acid, lysine, glutamic acid.

A final aspect of the invention relates to a method for altering thethermostability and/or of altering the specific activity of a parentglucoamylase by making one or more mutation(s) in one or more of thefollowing position(s) or region(s) corresponding to the position(s) orregion(s) of the amino acid sequence shown in NO: 2 of WO 00/04136:

-   a) said mutation(s) comprising one or more insertion(s) in: Region:    1-35, Region: 40-58, Region: 60-62, Region: 73-80, Position: 93,    Region: 95-101, Region: 103-121, Region: 123-124, Region: 126-127,    Region: 170-175, Region: 177-184, Region: 200-212, Region: 234-246,    Region: 287-312, Region: 314-319, Region: 334-339, Position: 341,    Region: 354-356, Position: 358, Region: 360-374, Region: 388-392,    Position: 94, Region: 396401, Region: 403-407, Region: 409414,    Region: 445449, Region: 452-467, Region: 469470, and/or in a    corresponding position or region in a homologous glucoamylase which    displays at least 60% homology with the amino acid sequences shown    in SEQ ID NO: 2 of WO 00/04136, or-   b) said mutation(s) comprising one or more substitution(s),    insertion(s) and/or deletion(s) in: Region: 36-39, Region: 63-65,    Position 67, Region: 69-71, Region: 81-92, Region: 128-169, Region:    85-188, Region: 190-199, Region: 213-222, Region: 224-226, Region:    228-233, Region: 247-271, Region: 273-286, Region: 320-333, Region:    343-344, Region: 346-347, Region: 349-351, Region: 375-378, Region:    380-385, Position: 387, Position: 415, Region: 417424, Position:    426, Region: 428443, Region: 471-485, Region: 487-489, Region:    491493, Region: 495-616, and/or in a corresponding position or    region in a homologous glucoamylase which displays at least 60%    homology with the amino acid sequence shown in SEQ ID NO: 2 of WO    00/04136, except the following amino acid substitutions: A39V,    P128S, G137A, G139A, D153N, S185H, G251A, D257E, E259D, E259Q,    C320A, D375C, G383A, W417F, S431C, A435P, S436P, W437F, A442T,    A471C, A479C, T480C, P481C, A495T and A495P.

Preferred embodiments of the final aspect are identical to those of thefirst aspect and relate to one or more mutation(s) in specificposition(s) corresponding to specific position(s) of SEQ ID NO: 2 in WO00/04136, as shown above.

Homology (Identity)

The homology referred to above of the parent glucoamylase is determinedas the degree of identity between two protein sequences indicating aderivation of the first sequence from the second. The homology maysuitably be determined by means of computer programs known in the artsuch as GAP provided in the GCG program package (Program Manual for theWisconsin Package, Version 8, August 1994, Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch,C. D., (1970), Journal of Molecular Biology, 48, p. 443-453). Using Gapwith the following settings for polypeptide sequence comparison: Gapcreation penalty of 3.0 and Gap extension penalty of 0.1, the maturepart of a polypeptide encoded by an analogous DNA sequence of theinvention exhibits a degree of identity preferably of at least 60%, suchas 70%, at least 80%, at least 90%, more preferably at least 95%, morepreferably at least 97%, and most preferably at least 99% with themature part of the amino acid sequence shown in SEQ ID NO: 2 of WO00/04136.

Preferably, the parent glucoamylase comprise the amino acid sequences ofSEQ ID NO: 2 in WO 00/04136; or allelic variants thereof; or fragmentsthereof that have glucoamylase activity.

A fragment of the amino acid sequence shown in SEQ ID NO: 2 of WO00/04136 is a polypeptide which has one or more amino acids deleted fromthe amino and/or carboxyl terminus of this amino acid sequence. Forinstance, the AMG G2 (SEQ ID NO: 2 of WO 00/04136) is a fragment of theAspergillus niger G1 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p.1097-1102) having glucoamylase activity. An allelic variant denotes anyof two or more alternative forms of a gene occupying the samechromosomal locus. Allelic variation arises naturally through mutation,and may result in polymorphism within populations. Gene mutations can besilent (no change in the encoded polypeptide) or may encode polypeptideshaving altered amino acid sequences. An allelic variant of a polypeptideis a polypeptide encoded by an allelic variant of a gene.

The amino acid sequences of homologous parent glucoamylases may differfrom the amino acid sequence of SEQ ID NO: 2 in WO 00/04136 by aninsertion or deletion of one or more amino acid residues and/or thesubstitution of one or more amino acid residues by different amino acidresidues. Preferably, amino acid changes are of a minor nature, that isconservative amino acid substitutions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof one to about 30 amino acids; small amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue; a small linkerpeptide of up to about 20-25 residues; or a small extension thatfacilitates purification by changing net charge or another function,such as a poly-histidine tract, an antigenic epitope or a bindingdomain.

In another embodiment, the isolated parent glucoamylase is encoded by anucleic acid sequence which hybridises under very low stringencyconditions, preferably low stringency conditions, more preferably mediumstringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with a nucleic acid probewhich hybridises under the same conditions with (i) the nucleic acidsequence of SEQ ID NO: 1 in WO 00/04136, (ii) the cDNA sequence of SEQID NO:1 in WO 00/04136, (iii) a sub-sequence of (i) or (ii), or (iv) acomplementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch,and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2dedition, Cold Spring Harbor, N.Y.). The sub-sequence of SEQ ID NO: 1 inWO 00/04136 may be at least 100 nucleotides or preferably at least 200nucleotides. Moreover, the sub-sequence may encode a polypeptidefragment which has glucoamylase activity. The parent polypeptides mayalso be allelic variants or fragments of the polypeptides that haveglucoamylase activity.

The nucleic acid sequence of SEQ ID NO: 1 in WO 00/04136 or asubsequence thereof, as well as the amino acid sequence of SEQ ID NO: 2in WO 00/04136, or a fragment thereof, may be used to design a nucleicacid probe to identify and clone DNA encoding polypeptides havingglucoamylase activity, from strains of different genera or speciesaccording to methods well known in the art. In particular, such probescan be used for hybridization with the genomic or cDNA of the genus orspecies of interest, following standard Southern blotting procedures, inorder to identify and isolate the corresponding gene therein. Suchprobes can be considerably shorter than the entire sequence, but shouldbe at least 15, preferably at least 25, and more preferably at least 35nucleotides in length. Longer probes can also be used. Both DNA and RNAprobes can be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA that hybridizes with the probes described aboveand which encodes a polypeptide having glucoamylase. Genomic or otherDNA from such other organisms may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilised on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that is homologous with SEQ ID NO: 1 inWO 00/04136, or sub-sequences thereof, the carrier material is used in aSouthern blot. For purposes of the present invention, hybridisationindicates that the nucleic acid sequence hybridises to a nucleic acidprobe corresponding to the nucleic acid sequence shown in SEQ ID NO: 1of WO 00/04136, its complementary strand, or a sub-sequence thereof,under very low to very high stringency conditions. Molecules to whichthe nucleic acid probe hybridises under these conditions are detectedusing X-ray film.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridisation, and washing post-hybridization at 5° C. to 10° C. belowthe calculated T_(m) using the calculation according to Bolton andMcCarthy (1962, Proceedings of the National Academy of Sciences USA48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40,1× Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasicphosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standardSouthern blotting procedures.

For short probes, which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

The present invention also relates to isolated nucleic acid sequencesproduced by (a) hybridising a DNA under very low, low, medium,medium-high, high, or very high stringency conditions with the sequenceof SEQ ID NO:1 in WO 00/04136, or its complementary strand, or asub-sequence thereof; and (b) isolating the nucleic acid sequence. Thesub-sequence is preferably a sequence of at least 100 nucleotides suchas a sequence, which encodes a polypeptide fragment, which hasglucoamylase activity.

Contemplated parent glucoamylases have at least 20%, preferably at least40%, more preferably at least 60%, even more preferably at least 80%,even more preferably at least 90%, and most preferably at least 100% ofthe glucoamylase activity of the mature polypeptide of SEQ ID NO: 2 inWO 00/04136.

In a preferred embodiment the variant of the invention has improvedthermal stability and/or increased specific activity, preferably withinthe temperature interval from about 60-80° C., preferably 63-75° C.,preferably at a pH of 4-5, in particular 4.24.7, using maltodextrin asthe substrate.

In another preferred embodiment a variant of the invention is used for,e.g., alcohol fermentation.

In a preferred embodiment the parent glucoamylase is the Aspergillusniger G1 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102.The parent glucoamylase may be a truncated glucoamylase, e.g., the AMGG2 glucoamylase.

Cloning a DNA Sequence Encoding a Parent Glucoamylase

The DNA sequence encoding a parent glucoamylase may be isolated from anycell or microorganism producing the glucoamylase in question, usingvarious methods well known in the art. First, a genomic DNA and/or cDNAlibrary should be constructed using chromosomal DNA or messenger RNAfrom the organism that produces the glucoamylase to be studied. Then, ifthe amino acid sequence of the glucoamylase is known, labeledoligonucleotide probes may be synthesized and used to identifyglucoamylase-encoding clones from a genomic library prepared from theorganism in question. Alternatively, a labelled oligonucleotide probecontaining sequences homologous to another known glucoamylase gene couldbe used as a probe to identify glucoamylase-encoding clones, usinghybridization and washing conditions of very low to very highstringency. This is described above.

Yet another method for identifying glucoamylase-encoding clones wouldinvolve inserting fragments of genomic DNA into an expression vector,such as a plasmid, transforming glucoamylase-negative bacteria with theresulting genomic DNA library, and then plating the transformed bacteriaonto agar containing a substrate for glucoamylase (i.e., maltose),thereby allowing clones expressing the glucoamylase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g. the phosphoroamiditemethod described S. L. Beaucage and M. H. Caruthers, (1981), TetrahedronLetters 22, p. 1859-1869, or the method described by Matthes et al.,(1984), EMBO J. 3, p. 801-805. In the phosphoroamidite method,oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer,purified, annealed, ligated and cloned in appropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate, the fragments corresponding to various parts of the entireDNA sequence), in accordance with standard techniques. The DNA sequencemay also be prepared by polymerase chain reaction (PCR) using specificprimers, for instance as described in U.S. Pat. No. 4,683,202 or R. K.Saiki et al., (1988), Science 239, 1988, pp. 487-491.

Site-Directed Mutagenesis

Once a glucoamylase-encoding DNA sequence has been isolated, anddesirable sites for mutation identified, mutations may be introducedusing synthetic oligonucleotides. These oligonucleotides containnucleotide sequences flanking the desired mutation sites. In a specificmethod, a single-stranded gap of DNA, the glucoamylase-encodingsequence, is created in a vector carrying the glucoamylase gene. Thenthe synthetic nucleotide, bearing the desired mutation, is annealed to ahomologous portion of the single-stranded DNA. The remaining gap is thenfilled in with DNA polymerase I (Klenow fragment) and the construct isligated using T4 ligase. A specific example of this method is describedin Morinaga et al., (1984), Biotechnology 2, p. 646-639. U.S. Pat. No.4,760,025 discloses the introduction of oligonucleotides encodingmultiple mutations by performing minor alterations of the cassette.However, an even greater variety of mutations can be introduced at anyone time by the Morinaga method, because a multitude ofoligonucleotides, of various lengths, can be introduced.

Another method for introducing mutations into glucoamylase-encoding DNAsequences is described in Nelson and Long, (1989), AnalyticalBiochemistry 180, p. 147-151. It involves the 3-step generation of a PCRfragment containing the desired mutation introduced by using achemically synthesized DNA strand as one of the primers in the PCRreactions. From the PCR-generated fragment, a DNA fragment carrying themutation may be isolated by cleavage with restriction endonucleases andreinserted into an expression plasmid.

Further, Sierks. et al., (1989), Protein Eng., 2, 621-625; Sierks etal., (1990), Protein Eng. vol. 3, 193-198; also describes site-directedmutagenesis in an Aspergillus glucoamylase.

Random Mutagenesis

Random mutagenesis is suitably performed either as localized orregion-specific random mutagenesis in at least three parts of the genetranslating to the amino acid sequence shown in question, or within thewhole gene.

The random mutagenesis of a DNA sequence encoding a parent glucoamylasemay be conveniently performed by use of any method known in the art.

In relation to the above, a further aspect of the present inventionrelates to a method for generating a variant of a parent glucoamylase,wherein the variant exhibits increased thermal stability relative to theparent, the method comprising:

-   -   (a) subjecting a DNA sequence encoding the parent glucoamylase        to random mutagenesis,    -   (b) expressing the mutated DNA sequence obtained in step (a) in        a host cell, and    -   (c) screening for host cells expressing a glucoamylase variant        which has an altered property (i.e. thermal stability) relative        to the parent glucoamylase.

Step (a) of the above method of the invention is preferably performedusing doped primers, as described in the working examples herein (videinfra).

For instance, the random mutagenesis may be performed by use of asuitable physical or chemical mutagenizing agent, by use of a suitableoligonucleotide, or by subjecting the DNA sequence to PCR generatedmutagenesis. Furthermore, the random mutagenesis may be performed by useof any combination of these mutagenizing agents. The mutagenizing agentmay, e.g., be one which induces transitions, transversions, inversions,scrambling, deletions, and/or insertions.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) ir-radiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues. When such agents are used, themutagenesis is typically performed by incubating the DNA sequenceencoding the parent enzyme to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions for themutagenesis to take place, and selecting for mutated DNA having thedesired properties.

When the mutagenesis is performed by the use of an oligonucleotide, theoligonucleotide may be doped or spiked with the three non-parentnucleotides during the synthesis of the oligonucleotide at thepositions, which are to be changed. The doping or spiking may be done sothat codons for unwanted amino acids are avoided. The doped or spikedoligonucleotide can be incorporated into the DNA encoding theglucoamylase enzyme by any published technique, using, e.g., PCR, LCR orany DNA polymerase and ligase as deemed appropriate.

Preferably, the doping is carried out using “constant random doping”, inwhich the percentage of wild-type and mutation in each position ispredefined. Furthermore, the doping may be directed toward a preferencefor the introduction of certain nucleotides, and thereby a preferencefor the introduction of one or more specific amino acid residues. Thedoping may be made, e.g., so as to allow for the introduction of 90%wild type and 10% mutations in each position. An additionalconsideration in the choice of a doping scheme is based on genetic aswell as protein-structural constraints. The doping scheme may be made byusing the DOPE program which, inter alia, ensures that introduction ofstop codons is avoided.

When PCR-generated mutagenesis is used, either a chemically treated ornon-treated gene encoding a parent glucoamylase is subjected to PCRunder conditions that increase the mis-incorporation of nucleotides(Deshler 1992; Leung et al., Technique, Vol.1, 1989, pp. 11-15).

A mutator strain of E. coli (Fowler et al., Molec. Gen. Genet., 133,1974, pp. 179-191), S. cereviseae or any other microbial organism may beused for the random mutagenesis of the DNA encoding the glucoamylase by,e.g., transforming a plasmid containing the parent glycosylase into themutator strain, growing the mutator strain with the plasmid andisolating the mutated plasmid from the mutator strain. The mu-ta-tedplasmid may be subsequently transformed into the expression organism.

The DNA sequence to be mutagenized may be conveniently present in agenomic or cDNA library prepared from an organism expressing the parentglucoamylase. Alternatively, the DNA sequence may be present on asuitable vector such as a plasmid or a bacteriophage, which as such maybe incubated with or other-wise exposed to the mutagenising agent. TheDNA to be mutagenized may also be present in a host cell either by beingintegrated in the genome of said cell or by being present on a vectorharboured in the cell. Finally, the DNA to be mutagenized may be inisolated form. It will be understood that the DNA sequence to besubjected to random mutagenesis is preferably a cDNA or a genomic DNAsequence.

In some cases it may be convenient to amplify the mutated DNA sequenceprior to performing the expression step b) or the screening step c).Such amplification may be performed in accordance with methods known inthe art, the presently preferred method being PCR-generatedamplification using oligonucleotide primers prepared on the basis of theDNA or amino acid sequence of the parent enzyme.

Subsequent to the incubation with or exposure to the mutagenising agent,the mutated DNA is expressed by culturing a suitable host cell carryingthe DNA sequence under conditions allowing expression to take place. Thehost cell used for this purpose may be one which has been transformedwith the mutated DNA sequence, optionally present on a vector, or onewhich carried the DNA sequence encoding the parent enzyme during themutagenesis treatment. Examples of suitable host cells are thefollowing: gram positive bacteria such as Bacillus subtilis, Bacilluslicheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium, Bacillus thuringiensis, Streptomyces lividans orStreptomyces murinus; and gram-negative bacteria such as E. coli.

The mutated DNA sequence may further comprise a DNA sequence encodingfunctions permitting expression of the mutated DNA sequence.

Localized Random Mutagenesis

The random mutagenesis may be advantageously localized to a part of theparent glucoamylase in question. This may, e.g., be advantageous whencertain regions of the enzyme have been identified to be of particularimportance for a given property of the enzyme, and when modified areexpected to result in a variant having improved properties. Such regionsmay normally be identified when the tertiary structure of the parentenzyme has been elucidated and related to the function of the enzyme.

The localized, or region-specific, random mutagenesis is convenientlyperformed by use of PCR generated mutagenesis techniques as describedabove or any other suitable technique known in the art. Alternatively,the DNA sequence encoding the part of the DNA sequence to be modifiedmay be isolated, e.g., by insertion into a suitable vector, and saidpart may be subsequently subjected to mutagenesis by use of any of themutagenesis methods discussed above.

Alternative methods for providing variants of the invention include geneshuffling e.g. as described in WO 95/22625 (from Affymax TechnologiesN.V.) or in WO 96/00343 (from Novo Nordisk A/S).

Expression of Glucoamylase Variants

According to the invention, a DNA sequence encoding the variant producedby methods described above, or by any alternative methods known in theart, can be expressed, in enzyme form, using an expression vector whichtypically includes control sequences encoding a promoter, operator,ribosome binding site, translation initiation signal, and, optionally, arepressor gene or various activator genes.

Expression Vector

The recombinant expression vector carrying the DNA sequence encoding aglucoamylase variant of the invention may be any vector which mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. The vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated. Examples ofsuitable expression vectors include pMT838.

Promoter

In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence, whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell.

Examples of suitable promoters for directing the transcription of theDNA sequence encoding a glucoamylase variant of the invention,especially in a bacterial host, are the promoter of the lac operon of E.coli, the Streptomyces coelicolor agarase gene dagA promoters, thepromoters of the Bacillus licheniformis alpha-amylase gene (amyL), thepromoters of the Bacillus stearothermophilus maltogenic amylase gene(amyM), the promoters of the Bacillus amyloliquefaciens alpha-amylase(amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc.For transcription in a fungal host, examples of useful promoters arethose derived from the gene encoding A. oryzae TAKA amylase, the TPI(triose phosphate isomerase) promoter from S. cerevisiae (Alber et al.(1982), J. Mol. Appl. Genet 1, p. 419-434, Rhizomucor miehei asparticproteinase, A. niger neutral alpha-amylase, A. niger acid stablealpha-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A.oryzae alkaline protease, A. oryzae triose phosphate isomerase or A.nidulans acetamidase.

Expression Vector

The expression vector of the invention may also comprise a suitabletranscription terminator and, in eukaryotes, polyadenylation sequencesoperably connected to the DNA sequence encoding the alpha-amylasevariant of the invention. Termination and polyadenylation sequences maysuitably be derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question. Examples of such sequences arethe origins of replication of plasmids pUC19, pACYC177, pUB110, pE194,pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argB, niaD and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g. as described in WO 91/17243.

The procedures used to ligate the DNA construct of the inventionencoding a glucoamylase variant, the promoter, terminator and otherelements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989).

Host Cells

The cell of the invention, either comprising a DNA construct or anexpression vector of the invention as defined above, is advantageouslyused as a host cell in the recombinant production of a glucoamylasevariant of the invention. The cell may be transformed with the DNAconstruct of the invention encoding the variant, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g. by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

The cell of the invention may be a cell of a higher organism such as amammal or an insect, but is preferably a microbial cell, e.g., abacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are Gram positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gramnegative bacteria such asE.coli. The transformation of the bacteria may, for instance, beeffected by protoplast transformation or by using competent cells in amanner known per se.

The yeast organism may favorably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae.

The host cell may also be a filamentous fungus e.g. a strain belongingto a species of Aspergillus, most preferably Aspergillus oryzae orAspergillus niger, or a strain of Fusarium, such as a strain of Fusariumoxysporium, Fusarium graminearum (in the perfect state named Gribberellazeae, previously Sphaeria zeae, synonym with Gibberella roseum andGibberella roseum f. sp. cerealis), or Fusarium sulphureum (in theprefect state named Gibberella puricaris, synonym with Fusariumtrichothecioides, Fusarium bactridioides, Fusarium sambucium, Fusariumroseum, and Fusarium roseum var. graminearum), Fusarium cerealis(synonym with Fusarium crokkwellnse), or Fusarium venenatum.

In a preferred embodiment of the invention the host cell is a proteasedeficient or protease minus strain.

This may for instance be the protease deficient strain Aspergillusoryzae JaL125 having the alkaline protease gene named “alp” deleted.This strain is described in WO 97/35956 (Novo Nordisk).

Filamentous fungi cells may be transformed by a process involvingprotoplast formation and transformation of the protoplasts followed byregeneration of the cell wall in a manner known per se. The use ofAspergillus as a host micro-organism is described in EP 238 023 (NovoNordisk AIS), the contents of which are hereby incorporated byreference.

Method of Producing a Glucoamylase Variant

In a yet further aspect, the present invention relates to a method ofproducing a glucoamylase variant of the invention, which methodcomprises cultivating a host cell under conditions conducive to theproduction of the variant and recovering the variant from the cellsand/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in question and obtaining expressionof the glucoamylase variant of the invention. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes (e.g. as described in catalogues of the American TypeCulture Collection).

The glucoamylase variant secreted from the host cells may convenientlybe recovered from the culture medium by well-known procedures, includingseparating the cells from the medium by centrifugation or filtration,and precipitating proteinaceous components of the medium by means of asalt such as ammonium sulphate, followed by the use of chromatographicprocedures such as ion exchange chromatography, affinity chromatography,or the like.

Starch Conversion

The present invention provides a method of using glucoamylase variantsof the invention for producing glucose and the like from starch.Generally, the method includes the steps of partially hydrolyzingprecursor starch in the presence of alpha-amylase and then furtherhydrolyzing the release of D-glucose from the non-reducing ends of thestarch or related oligo- and polysaccharide molecules in the presence ofglucoamylase by cleaving alpha-(1→4) and alpha-(1→6) glucosidic bonds.

The partial hydrolysis of the precursor starch utilizing alpha-amylaseprovides an initial breakdown of the starch molecules by hydrolyzinginternal alpha-(1→4)-linkages. In commercial applications, the initialhydrolysis using alpha-amylase is run at a temperature of approximately105° C. A very high starch concentration is processed, usually 30% to40% solids. The initial hydrolysis is usually carried out for fiveminutes at this elevated temperature. The partially hydrolyzed starchcan then be transferred to a second tank and incubated for approximately1-2 hour at a temperature of 85° to 98° C. to derive a dextroseequivalent (D.E.) of 10 to 15.

The step of further hydrolyzing the release of D-glucose from thenon-reducing ends of the starch or related oligo- and polysaccharidesmolecules in the presence of glucoamylase is normally carried out in aseparate tank at a reduced temperature between 30° and 62° C. Preferablythe temperature of the substrate liquid is dropped to between 550 and60° C. The pH of the solution is dropped from about 5.5 to 6.5 to arange between 3 and 5.5. Preferably, the pH of the solution is 4 to 4.5.The glucoamylase is added to the solution and the reaction is carriedout for 24-72 hours, preferably 3648 hours.

By using a thermostable glucoamylase variant of the inventionsaccharification processes may be carried out at a higher temperaturethan traditional batch saccharification processes. According to theinvention saccharification may be carried out at temperatures in therange from above 60-80° C., preferably 63-75° C. This apply both fortraditional batch processes (described above) and for continuoussaccharification processes.

Actually, continuous saccharification processes including one or moremembrane separation steps, i.e. filtration steps, must be carried out attemperatures of above 60° C. to be able to maintain a reasonably highflux over the membrane or to minimize microbial contamination.Therefore, the thermostable variants of the invention provides thepossibility of carrying out large scale continuous saccharificationprocesses at a fair price and/or at a lower enzyme protein dosage withina period of time acceptable for industrial saccharification processes.According to the invention the saccharification time may even beshortened.

The activity of the glucoamylase variant (e.g. AMG variant) of theinvention is generally substantially higher at temperatures between 60°C.-80° C. than at the traditionally used temperature between 30-60° C.Therefore, by increasing the temperature at which the glucoamylaseoperates the saccharification process may be carried out within ashorter period of time.

Further, by improving the thermal stability the T_(1/2) (half-time, asdefined in the “Materials and Methods” section) is improved. As thethermal stability of the glucoamylase variants of the invention isimproved a minor amount of glucoamylase need to be added to replace theglucoamylase being inactivated during the saccharification process. Moreglucoamylase is maintained active during saccharification processaccording to the present invention. Furthermore, the risk of microbialcontamination is also reduced when carrying the saccharification processat temperature above 63° C.

The glucose yield from a typical saccharification trial withglucoamylase, acid amylase and pullulanase is 95.5-96.5%. The remainingcarbohydrates typically consists of 1% maltose, 1.5-2% isomaltose and1-1.5% higher oligosacharides. The disaccharides are produced since theglucoamylase at high concentrations of glucose and high dry-solid levelshas a tendency to form reversion products.

A glucoamylase with an increased specific activity towards saccharidespresent in the solution after liquefaction and saccharides formed duringsaccharification would be an advantage as a reduced enzyme proteindosage or a shorter process time then could be used. In general, theglucoamylase has a preference for substrates consisting of longersaccharides compared to short chain saccharides and the specificactivity towards e.g. maltoheptaose is therefore approximately 6 timeshigher than towards maltose. An increased specific activity towardsshort chain saccharides such as maltose (without reducing the activitytowards oligosaccharides) would therefore also permit using a lowerenzyme dosage and/or shorter process time.

Furthermore, a higher glucose yield can be obtained with a glucoamylasevariant with an increased alpha-1,4 hydrolytic activity (if thealpha-1,6 activity is unchanged or even decreased), since a reducedamount of enzyme protein is being used, and alpha-1,6 reversion productformation therefore is decreased (less isomaltose).

The specific activity may be measured using the method described in the“Materials & Methods” section at 37° C. or 60° C.

Example of saccharification process wherein the glucoamylase variants ofthe invention may be used include the processes described in JP3-224493; JP 1-191693; JP 62-272987; EP 452,238, and WO 99/27124 (allreferences are hereby incorporated by reference).

In a further aspect the invention relates to a method of saccharifying aliquefied starch solution, comprising the steps

-   -   (i) a saccharification step during which step one or more        enzymatic saccharification stages takes place, and the        subsequent step of    -   (ii) one or more high temperature membrane separation steps        wherein the enzymatic saccharification is carried out using a        thermostable glucoamylase variant of the invention.

The glucoamylase variant(s) of the invention may be used in the presentinventive process in combination with an enzyme that hydrolyzes onlyα-(1

6)-glucosidic bonds in molecules with at least four glucosyl residues.Preferentially, the glucoamylase variant of the invention can be used incombination with pullulanase or isoamylase. The use of isoamylase andpullulanase for debranching, the molecular properties of the enzymes,and the potential use of the enzymes with glucoamylase is set forth inG. M. A. van Beynum et al., Starch Conversion Technology, Marcel Dekker,New York, 1985, 101-142.

In a further aspect the invention relates to the use of a glucoamylasevariant of the invention in a starch conversion process.

Further, the glucoamylase variant of the invention may be used in acontinuous starch conversion process including a continuoussaccharification step.

The glucoamylase variants of the invention may also be used inimmobilised form. This is suitable and often used for producingspeciality syrups, such as maltose syrups, and further for the raffinatestream of oligosaccharides in connection with the production of fructosesyrups.

The glucoamylase of the invention may also be used in a process forproducing ethanol for fuel or beverage or may be used in a fermentationprocess for producing organic compounds, such as citric acid, ascorbicacid, lysine, glutamic acid.

Materials & Methods

Materials:

Enzymes:

AMG G1: Aspergillus niger glucoamylase G1 disclosed in Boel et al.(1984), EMBO J. 3 (5), 1097-1102, and SEQ ID NO: 13 of WO 00/04136,available from Novo Nordisk.

AMG G2: Truncated Aspergillus niger glucoamylase G1 shown in SEQ ID NO:2 in WO 00/04136 (available from Novo Nordisk)

Solutions:

Buffer: 0.05M sodium acetate (6.8 g in 1 l milli-Q-water), pH 4.5

Stop solution: 0.4M NaOH

GOD-perid, 124036, Boehringer Mannheim

Substrate:

Maltose: 29 mM (Ig maltose in 100 ml 50 mM sodium acetate, pH 4.5)(Sigma)

Maltoheptaose: 10 mM, 115 mg/10 ml (Sigma)

Host Cell:

A. oryzae JaL 125: Aspergillus oryzae IFO 4177 available from Institutefor Fermention, Osaka; 17-25 Juso Hammachi 2-Chome Yodogawa-ku, Osaka,Japan, having the alkaline protease gene named “alp” (described byMurakami K et al., (1991), Agric. Biol. Chem. 55, p. 2807-2811) deletedby a one step gene replacement method (described by G. May in “AppliedMolecular Genetics of Filamentous Fungi” (1992), p. 1-25. Eds. J. R.Kinghom and G. Turner; Blackie Academic and Professional), using the A.oryzae pyrG gene as marker. Strain JaL 125 is further disclosed in WO97/35956 (Novo Nordisk).

Microorganisms:

Strain: Saccharomyces cerevisiae YNG318: MATαleu2-Δ2 ura3-52 his4-539pep4-Δ1 [cir+]

Plasmids:

pCAMG91: See FIG. 1 of WO 00/04136. Plasmid comprising the Aspergillusniger G1 glucoamylase (AMG G1). The construction of pCAMG91 is describedin Boel et al. (1984), EMBO J. 3 (7) p.1581-1585.

pMT838: Plasmid encoding the truncated Aspergillus niger glucoamylase G2(SEQ ID NO: 2 in WO 00/04136).

pJSO026 (S. cerevisiae expression plasmid) (J. S. Okkels, (1996) “AURA3-promoter deletion in a pYES vector increases the expression levelof a fungal lipase in Saccharomyces cerevisiae. Recombinant DNABiotechnology III: The Integration of Biological and EngineeringSciences, vol. 782 of the Annals of the New York Academy of Sciences)More specifically, the expression plasmid pJSO37, is derived from pYES2.0 by replacing the inducible GALL-promoter of pYES 2.0 with theconstitutively expressed TPI (triose phosphate isomerase)-promoter fromSaccharomyces cerevisiae (Albert and Karwasaki, (1982), J. Mol. ApplGenet., 1, 419-434), and deleting a part of the URA3 promoter.

Methods:

Transformation of Saccharomyces cerevisiae YNG318

The DNA fragments and the opened vectors are mixed and transformed intothe yeast 30 Saccharomyces cerevisiae YNG318 by standard methods.

Determining Specific Activity as k_(cat) (sec.⁻¹).

750 microL substrate (1% maltose, 50 mM Sodium acetat, pH 4.3) isincubated 5 minutes at selected temperature, such as 37° C. or 60° C.

50 microL enzyme diluted in sodium acetate is added.

Aliquots of 100 microL are removed after 0, 3, 6, 9 and 12 minutes andtransferred to 100 microL 0.4 M Sodium hydroxide to stop the reaction. Ablank is included.

20 microL is transferred to a Micro titre plates and 200 microLGOD-Perid solution is added. Absorbance is measured at 650 nm after 30minutes incubation at room temperature.

Glucose is used as standard and the specific activity is calculated ask_(cat) (sec.⁻¹).

Determination of AGU Activity and as AGU/mg

One Novo Amyloglucosidase Unit (AGU) is defined as the amount of enzymewhich hydrolyzes 1 micromole maltose per minute at 37° C. and pH 4.3. Adetailed description of the analytical method (AEL-SM-0131) is availableon request from Novo Nordisk.

The activity is determined as AGU/ml by a method modified after(AEL-SM-0131) using the Glucose GOD-Perid kit from Boehringer Mannheim,124036. Standard: AMG-standard, batch 7-1195, 195 AGU/ml.

375 microL substrate (1% maltose in 50 mM Sodium acetate, pH 4.3) isincubated 5 minutes at 37° C. 25 microL enzyme diluted in sodium acetateis added. The reaction is stopped after 10 minutes by adding 100 microL0.25 M NaOH. 20 microL is transferred to a 96 well microtitre plate and200 microL GOD-Perid solution is added. After 30 minutes at roomtemperature, the absorbance is measured at 650 nm and the activitycalculated in AGU/ml from the AMG-standard.

The specific activity in AGU/mg is then calculated from the activity(AGU/ml) divided with the protein concentration (mg/ml).

Transformation of Aspergillus oryzae (General Procedure)

100 ml of YPD (Sherman et al., (1981), Methods in Yeast Genetics, ColdSpring Harbor Laboratory) are inoculated with spores of A. oryzae andincubated with shaking for about 24 hours. The mycelium is harvested byfiltration through miracloth and washed with 200 ml of 0.6 M MgSO₄. Themycelium is suspended in 15 ml of 1.2 M MgSO₄, 10 mM NaH₂PO₄, pH 5.8.The suspension is cooled on ice and 1 ml of buffer containing 120 mg ofNovozym™ 234 is added. After 5 min., 1 ml of 12 mg/ml BSA (Sigma typeH25) is added and incubation with gentle agitation continued for 1.5-2.5hours at 37 C until a large number of protoplasts is visible in a sampleinspected under the microscope.

The suspension is filtered through miracloth, the filtrate transferredto a sterile tube and overlayed with 5 ml of 0.6 M sorbitol, 100 mMTris-HCl, pH 7.0. Centrifugation is performed for 15 min. at 1000 g andthe protoplasts are collected from the top of the MgSO₄ cushion. 2volumes of STC (1.2 M sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mM CaCl₂) areadded to the protoplast suspension and the mixture is centrifugated for5 min. at 1000 g. The protoplast pellet is resuspended in 3 ml of STCand repelleted. This is repeated. Finally, the protoplasts areresuspended in 0.2-1 ml of STC. 100 microL of protoplast suspension aremixed with 5-25 μg of p3SR2 (an A. nidulans amdS gene carrying plasmiddescribed in Hynes et al., Mol. and Cel. Biol., Vol. 3, No. 8,1430-1439, August 1983) in 10 microL of STC. The mixture is left at roomtemperature for 25 min. 0.2 ml of 60% PEG 4000 (BDH 29576), 10 mM CaCl₂and 10 mM Tris-HCl, pH 7.5 is added and carefully mixed (twice) andfinally 0.85 ml of the same solution are added and carefully mixed. Themixture is left at room temperature for 25 min., spun at 2.500 g for 15min. and the pellet is resuspended in 2 ml of 1.2 M sorbitol. After onemore sedimentation the protoplasts are spread on minimal plates (Cove,(1966), Biochem. Biophys. Acta 113, 51-56) containing 1.0 M sucrose, pH7.0, 10 mM acetamide as nitrogen source and 20 mM CsCl to inhibitbackground growth. After incubation for 4-7 days at 37° C. spores arepicked, suspended in sterile water and spread for single colonies. Thisprocedure is repeated and spores of a single colony after the secondre-isolation are stored as a defined transformant.

Fed Batch Fermentation

Fed batch fermentation is performed in a medium comprising maltodextrinas a carbon source, urea as a nitrogen source and yeast extract. The fedbatch fermentation is performed by inoculating a shake flask culture ofA. oryzae host cells in question into a medium comprising 3.5% of thecarbon source and 0.5% of the nitrogen source. After 24 hours ofcultivation at pH 5.0 and 34° C. the continuous supply of additionalcarbon and nitrogen sources are initiated. The carbon source is kept asthe limiting factor and it is secured that oxygen is present in excess.The fed batch cultivation is continued for 4 days, after which theenzymes can be recovered by centrifugation, ultrafiltration, dearfiltration and germ filtration.

Purification

The culture broth is filtrated and added ammoniumsulphate (AMS) to aconcentration of 1.7 M AMS and pH is adjusted to pH 5. Precipitatedmaterial is removed by centrifugation and the solution containingglucoamylase activity is applied on a Toyo Pearl Butyl column previouslyequilibrated in 1.7 M AMS, 20 mM sodium acetate, pH 5. Unbound materialis washed out with the equilibration buffer. Bound proteins are elutedwith 10 mM sodium acetate, pH 4.5 using a linear gradient from 1.7-0 MAMS over 10 column volumes. Glucoamylase containing fractions arecollected and dialysed against 20 mM sodium acetate, pH 4.5. Thesolution was then applied on a Q sepharose column, previouslyequilibrated in 10 mM piperazin, Sigma, pH 5.5. Unbound material iswashed out with the equilibration buffer. Bound proteins are eluted witha linear gradient of 0-0.3 M Sodium chloride in 10 mM piperazin, pH 5.5over 10 column volumes. Glucoamylase containing fractions are collectedand the purity was confirmed by SDS-PAGE.

Construction of pAMGY

The pAMGY vector was constructed as follows: The lipase gene in pJSO026was replaced by the AMG gene, which was PCR amplified with the forwardprimer; FG2: 5′-CAT CCC CAG GAT CCT TAC TCA GCA ATG-3′ and the reverseprimer RG2: 5′-CTC AAA CGA CTC ACC AGC CTC TAG AGT-3′ using the templateplasmid pLAC103 containing the AMG gene. The pJSO026 plasmid wasdigested with XbaI and SmaI at 37° C. for 2 hours and the PCR ampliconwas blunt ended using the Klenow fragment and then digested with XbaI.The vector fragment and the PCR amplicon were ligated and transformedinto E. coli by electrotransformation. The resulting vector isdesignated pAMGY.

Construction of pLaC103

The A. niger AMGII cDNA clone (Boel et al., (1984), supra) is used assource for the construction of pLaC103 aimed at S. cerevisiae expressionof the GII form of AMG. The construction takes place In several steps,outlined below.

pT7-212 (EP37856/U.S. Pat. No. 5,162,498) is cleaved with XbaI,blunt-ended with Klenow DNA polymerase and dNTP. After cleavage withEcoRI the resulting vector fragment is purified from an agarosegel-electrophoresis and ligated with the 2.05 kb EcoR1-EcoRV fragment ofpBoel53, thereby recreating the XbaI site in the EcoRV end of the AMGencoding fragment in the resulting plasmid pG2x.

In order to remove DNA upstream of the AMG cds, and furnish the AMGencoding DNA with an appropriate restriction endonuclease recognitionsite, the following construct was made:

The 930 bp EcoRI-PstI fragment of p53 was isolated and subjected to AluIcleavage, the resulting 771 bp Alu-PstI fragment was ligated into pBR322with blunt-ended EcoRI site (see above) and cleaved with PstI In theresulting plasmid pBR-AMG′, the EcoRI site was recreated just 34 bp fromthe initiation codon of the AMG cds.

From pBR-AMG′ the 775 bp EcoRI-PstI fragment was isolated and joinedwith the 1151 bp PstI-XbaI fragment from pG2x in a ligation reactionincluding the XbaI-EcoRI vector fragment of pT7-212.

The resulting plasmid pT7GII was submitted to a BamHI cleavage inpresence of alkaline phosphatase followed by partial SphI cleavage afterinactivation of the phosphatase. From this reaction was the 2489 bpSphI-BamHI fragment, encompassing the S.c. TPI promoter linked to theAMGII cds.

The above fragment together with the 1052 bp BamHI fragment of pT7GIIwas ligated with the alkaline phosphatase treated vector fragment ofpMT743 (EP37856/U.S. Pat. No. 5,162,498), resulting from SphI-BamHIdigestion. The resulting plasmid is pLaC103.

Screening for Thermostable AMG Variants

The libraries are screened in the Filter Assay for Thermostability asdescribed in WO 00/04136.

General Method for Random Mutagenesis by Use of the DOPE Program

The random mutagenesis may be carried out by the following steps:

-   -   1. Select regions of interest for modification in the parent        enzyme,    -   2. Decide on mutation sites and non-mutated sites in the        selected region,    -   3. Decide on which kind of mutations should be carried out,        e.g., with respect to the desired stability and/or performance        of the variant to be constructed,    -   4. Select structurally reasonable mutations,    -   5. Adjust the residues selected by step 3 with regard to step 4.    -   6. Analyze by use of a suitable dope algorithm the nucleotide        distribution.    -   7. If necessary, adjust the wanted residues to genetic code        realism, e.g. taking into account constraints resulting from the        genetic code, e.g. in order to avoid introduction of stop        codons; the skilled person will be aware that some codon        combinations cannot be used in practice and will need to be        adapted    -   8. Make primers    -   9. Perform random mutagenesis by use of the primers    -   10. Select resulting glucoamylase variants by screening for the        desired improved properties.        Dope Algorithm

Suitable dope algorithms for use in step 6 are well known in the art.One such algorithm is described by Tomandl, D. et al., 1997, Journal ofComputer-Aided Molecular Design 11:29-38. Another algorithm is DOPE(Jensen, L J, Andersen, K V, Svendsen, A, and Kretzschmar, T (1998)Nucleic Acids Research 26:697-702).

Method Of Extracting Important Regions For Temperature Activity UsingMolecular Simulation. The X-ray structure and/or the model-buildstructure of the enzyme of interest, here AMG, are subjected tomolecular dynamics simulations. The molecular dynamics simulation aremade using the CHARMM (from Molecular simulations (MSI)) program orother suitable programs, e.g., DISCOVER (from MSI). The dynamics aremade in vacuum, or including crystal waters, or with the enzyme inquestion embedded in a suitable waters, e.g., a sphere or a box. Thesimulation are run for 300 picoseconds (ps) or more, e.g., 300-1200 ps.The isotropic fluctuations are extracted for the CA carbons of thestructures and comparison between the structures are made. More detailson how to get the isotropic fluctuations can be found in the CHARMMmanual (available from MSI) and hereby incorporated herein by reference.The molecular dynamics simulation can be carried out using standardcharges on the chargeable amino acids. For instance, Asp and Glu isnegatively charged and Lys and Arg are positively charged. Thiscondition resembles the medium pH of approximately 7.0. To analyze alower pH, titration of the molecule can be done to obtain the alteredpKa's of the normal titrateable residues within pH 2-10; Lys, Arg, Asp,Glu, Tyr and His. Also Ser, Thr and Cys are titrateable but are nottaking into account here. Here the altered charges due to the pH hasbeen described as all Arg, Lys negative at high pH, and all Asp, Glu areuncharged. This imitates a pH around 4 to 5 where the titration Asp andGlu normally takes place. Model building of the enzyme of interest canbe obtained by using the HOMOLOGY model in the MSI program package. Thecrystal structure of Aspergillus awamori variant X100 can be found in,e.g., 3GLY and 1DOG in the Brookhaven database.

EXAMPLES Example 1

Construction of AMG G2 Variants

Site-Directed Mutagenesis:

For the construction of variants of AMG G2 (SEQ ID NO: 2 of WO 00/04136,available from Novo Nordisk) the commercial kit, Chameleondouble-stranded, site-directed mutagenesis kit was used according to themanufacturer's instructions. The gene encoding the AMG G2 enzyme inquestion is located on pMT838 prepared by deleting the DNA between G2nt. 1362 and G2 nt. 1530 in plasmid pCAMG91 comprising the AMG G1 form.

In accordance with the manufacturer's instructions the ScaI site of theAmpicillin gene of pMT838 was changed to a MluI site by use of thefollowing primer:

Primer 7258: 5′p gaa tga ctt ggt tga cgc gtc acc agt cac (SEQ ID NO: 3of WO 00/04136)

(Thus changing the ScaI site found in the ampicillin resistance gene andused for cutting to a MluI site). The pMT838 vector comprising the AMGgene in question was then used as a template for DNA polymerase andprimer 7258 (SEQ ID NO: 3 of WO 00/04136) and primer 21401 (SEQ ID NO: 4of WO 00/04136). Primer 21401 (SEQ ID NO: 4 of WO 00/04136) was used asthe selection primer.

Primer 21401: 5′p gg gga tca tga tag gac tag cca tat taa tga agg gca tatacc acg cct tgg acc tgc gtt ata gcc (SEQ ID NO: 4 of WO 00/04136)

(Changes the ScaI site found in the AMG gene without changing the aminoacid sequence). The desired mutation (e.g., the introduction of acystein residue) is introduced into the AMG gene in question by additionof appropriate oligos comprising the desired mutation. The primer 107581was used to introduce T12P

Primer 107581: 5′ pgc aac gaa gcg ccc gtg gct cgt ac (SEQ ID NO: 5 of WO00/04136).

The mutations are verified by sequencing the whole gene. The plasmid wastransformed into A. oryzae using the method described above in the“Materials & Methods” section. The variant was fermented and purified asdescribed above in the “Materials & Methods” section.

Example 2

To improve the thermostability of the A. niger AMG, random mutagenesisin pre-selected region(s) was performed as shown in Example 2 of WO00/04136 which is incorporated herein by reference. Construction wasdone by localized random, doped mutagenesis, of A. niger AMG resultingin variants having altered thermostability compared to the parent AMGenzyme.

Example 3

A. niger AMG variants having improved thermostability compared to theparent enzyme. Construction, by PCR shuffling spiked with DNA oligos,was performed as described in 15 Example 3 of WO 00/04136, which isincorporated herein by reference.

Example 4

Specific Activity

AMG G2 variants were constructed as described above in Example 1. Thespecific activity as k_(cat) were measured on purified samples at pH4.3, 37° C., using maltose and maltohepatose as substrate as describedin the “Materials & Methods” section above. The specific activity asAGU/mg were also measured at pH 4.3, 37° C., using maltose as substrateas described in the “Materials & Methods” section above. Kcat (sec.-1)Variant Maltose Maltoheptaose AMG G2 (wt) 6.0 38 N110T 9.7 27.8 V111P12.0 43.2 S119P 6.2 44.0 G127A 21.0 40.0 G207N 30.5 36.3 Variant AGU/mgAMG G2 (wild type) 1.8 N110T 3.5 V111P 3.1 S119P 2.1 G127A 5.8 G207N 5.7L3N 2.3 S56A 2.6 A102* 2.5 D403S 2.2 I18V + T51S + S56A + V59T + L60A3.3 S119P + Y402F 2.7 S119P + I189T + Y223F + F227Y + Y402F 3.0

Example 5

Thermostability at 70° C.

An AMG G2 S119P variant was constructed using the approach described inExample 51.

The thermostability was determined as T_(1/2) using Method I, and as %residual activity after incubation for 30 minutes in 50 mM NaOAc, pH4.5, 70° C., 0.2 AGU/ml, as described in the “Material & Methods”section above. The result of the tests are listed in the Table below andcompared to the wild-type A. niger AMG G2. Residual T_(1/2) A. niger AMG(Enzyme) activity (%) (min.) S119P variant 22 17 wild-type (SEQ ID NO:2) 13 8

Example 6

Thermostability at 68° C.

AMG G2 variants were constructed using the approach described in Example3, except for variants nos. 1 and 2 in the Table below, which wereprepared by shuffling as described in WO 95/22625 (from AffymaxTechnologies N.V.). The thermostability was determined as T_(1/2) usingmethod I at 68° C. as described in the “Materials & Methods” section andcompared to the wild-type A. niger AMG G2 under the same conditions.Evaluation of variants were performed on culture broth after filtrationof the supernatants. T½ A. niger AMG G2 T½ (wild type) Variant (min)(min) 1 A246T + T72I 11.3 8.5 2 G447S + S119P 11.4 7.9 3 E408R + A425T +S465P + T494A 8.6 8.1 4 E408R + S386N 12.6 8.9 5 T2P 9.3 8.5 6 T2Q +A11P + S394R 10.7 8.5 7 T2H 9.5 8.9 8 A11E + E408R 12.7 9.3 9 T2M +N9A + T390R + D406N + L410R 10.7 8.5 10 A393R 17.7 8.4 11 T2R + S386R +A393R 14.1 8.4 12 A393R + L410R 14.7 7.9 13 A1V + L66R + Y402F + N427S +S486G 11.7 8.5 14 T2K + S30P + N427M + S444G + V470M 11.4 8.4

Thermostability at 70° C. on Purified Samples. Enzyme T½ (min) 15 AMG G2(wild type) 7.4 16 T2E + T379A + S386K + A393R 11.6 17 E408R + S386N10.2 18 T2Q + A11P + S394R 9.8 19 A1V + L66R + Y402F + N427S + S486G14.1 20 A393R 14.6 21 T2R + S386R + A393R 14.1 22 A393R + L410R 12.9 23Y402F 10.1

Example 7

Thermostability at 68° C.

AMG G2 variants were constructed by shuffling using the approachdescribed in Example 3 followed by shuffling of positive variants.

The thermostability was determined as T½ using method I at 68° C. asdescribed in the “Materials & Methods” section and compared to thewild-type A. niger AMG G2 under the same conditions. Evaluation ofvariants were performed on culture broth after filtration of thesupernatants. T½ A. niger AMG G2 (wild T½ type) Variant (min) (min) 24PLASD^(i) + V59A + A393R + T490A 27.2 6.8i = N-terminal extension

Example 8

Thermostability at 68° C.

AMG G2 variants were constructed using the approach described in Example3. The thermostability was determined as T½ using method I at 68° C. asdescribed in the “Materials & Methods” section and compared to thewild-type A. niger AMG G2 under the same conditions. Evaluation ofvariants were performed on culture broth after filtration of thesupernatants. T½ A. niger AMG G2 T½ wild-type Variant (min) (min) 25D357S + T360V + S371H 6.6 5.9 26 N313G + F318Y 8.9 5.9 27 S356P + S366T7.3 5.8 28 S340G + D357S + T360V + S386P 7.2 5.8

Example 9

Thermostability at 70° C.

An AMG G2 variants was constructed using the approach described inExample 1 and evaluated as semi-purified (filtration of culture brothfollowed by desalting on a G-25 column) samples.

The thermostability was determined as % residual activity using Method Iin 50 mM NaOAc, pH 4.5, 70° C., as described in the “Material & Methods”section above. The result of the test is listed in the Table below andcompared to the wild-type A. niger AMG G2. Enzyme T½ (min) 29 AMG G2(wild type) 7 30 Y402F + S411V 60 31 S119P + Y402F + S411V 115 32S119P + Y312Q + Y402F + T416H 50

Example 10

Thermostability at 70° C. in Presence of 30% Glucose

AMG G2 variants were constructed using the approach described in Example3.

The thermostability was determined as T/2 using method II at 70° C. asdescribed in the “Materials & Methods” section and compared to thewild-type A. niger AMG G2 under the same conditions. Enzyme T½ (hr) 33AMG G2 (wild type) 1.5 34 Y402F 2.5 35 A393R 4.0 36 T2R + S386R + A393R2.0 37 PLASD(N-terminal) + V59A + A393R + T490A 16.0

Example 11

Saccharification Performance of AMG Variants S119P+Y402F+S411V andPLASD(N-Terminal)+V59A+A393R+T490A, Respectively.

Saccharification performance of the AMG variants S119P+Y402F+S411V andPLASD(N-terminal)+V59A+A393R+T490A, respectively, both having improvedthermostability are tested at 70° C. as described below.

Reference enzyme is the wild-type A. niger AMG G2. Saccharification isrun under the following conditions: Substrate 10 DE Maltodextrin,approx. 30% DS (w/w) Temperature 70° C. Initial pH 4.3 (at 70° C.)Enzyme dosage 0.24 AGU/g DSSaccharification

The substrate for saccharification is made by dissolving maltodextrin(prepared from common corn) in boiling Milli-Q water and adjusting thedry substance to approximately 30% (w/w). pH is adjusted to 4.3.Aliquots of substrate corresponding to 15 g dry solids are transferredto 50 ml blue cap glass flasks and placed in a water bath with stirring.Enzymes are added and pH re-adjusted if necessary. The experiment is runin duplicate. Samples are taken periodically and analysed at HPLC fordetermination of the carbohydrate composition.

1-25 (Canceled.)
 26. A variant of a parent glucoamylase, wherein thevariant has at least 70% identity to the amino acid sequence publishedas SEQ ID NO: 2 in WO 00/04136, and wherein the variant comprises one ormore mutation(s) in position(s) or region(s) corresponding to thefollowing position(s) or region(s) in the amino acid sequence publishedas SEQ ID NO: 2 in WO 00/04136: a) said variant comprising one or moreinsertion(s) in: Region: 1-35, Region: 40-58, Region: 60-62, Region:73-80, Position: 93, Region: 95-101, Region: 103-121, Region: 123-124,Region: 126-127, Region: 170-175, Region: 177-184, Region: 200-212,Region: 234-246, Region: 287-312, Region: 314-319, Region: 334-339,Position: 341, Region: 354-356, Position: 358, Region: 360-374, Region:388-392, Position: 394, Region: 396-401, Region: 403407, Region:409-414, Region: 445-449, Region: 452-467, Region: 469-470, or b) saidvariant comprising one or more substitution(s), insertion(s) and/ordeletion(s) in: Region: 36-39, Region: 63-65, Position 67, Region:69-71, Region: 81-92, Region: 128-169, Region: 185-188, Region: 190-199,Region: 213-222, Region: 224-226, Region: 228-233, Region: 247-271,Region: 273-286, Region: 320-333, Region: 343-344, Region: 346-347,Region: 349-351, Region: 375-378, Region: 380-385, Position: 387,Position: 415, Region: 417-424, Position: 426, Region: 428-443, Region:471-485, Region: 487-489, Region: 491493, Region: 495-616, except thefollowing amino acid substitutions: A39V, P128S, G137A, G139A, D153N,S185H, G251A, D257E, E259D, E259Q, C320A, D375C, G383A, W417F, S431C,A435P, S436P, W437F, A442T, A471C, A479C, T480C, P481C, A495T and A495P.27. The variant of claim 26, wherein the variant has at least 80%identity to the amino acid sequence published as SEQ ID NO: 2 in WO00/04136.
 28. The variant of claim 26, wherein the variant has at least90% identity to the amino acid sequence published as SEQ ID NO: 2 in WO00/04136.
 29. The variant of claim 26, wherein the variant has at least95% identity to the amino acid sequence published as SEQ ID NO: 2 in WO00/04136.
 30. The variant of claim 26, wherein the variant has at least97% identity to the amino acid sequence published as SEQ ID NO: 2 in WO00/04136.
 31. The variant of claim 26, wherein the variant has at least99% identity to the amino acid sequence published as SEQ ID NO: 2 in WO00/04136.
 32. The variant of claim 26, wherein the parent glucoamylaseis the Aspergillus niger G1 glucoamylase.
 33. A DNA construct comprisinga DNA sequence encoding a glucoamylase variant according to claim 26.34. A recombinant expression vector which carries a DNA constructaccording to claim
 33. 35. A cell which is transformed with a DNAconstruct according to claim
 33. 36. The cell of claim 35, which is amicroorganism.
 37. The cell of claim 35, which is a bacterium or afungus.
 38. The cell of claim 35, which is a protease deficientAspergillus oryzae or Aspergillus niger.
 39. A process for convertingstarch or partially hydrolyzed starch into a syrup containing dextrose,said process including the step saccharifying starch hydrolyzate in thepresence of a glucoamylase variant of claim
 26. 40. The process of claim39, wherein the dosage of glucoamylase is present in the range from 0.05to 0.5 AGU per gram of dry solids.
 41. The process of claim 39,comprising saccharification of a starch hydrolyzate of at least 30percent by weight of dry solids.
 42. The process of claim 39, whereinthe saccharification is conducted in the presence of a debranchingenzyme selected from the group of pullulanase and isoamylase.
 43. Theprocess of claim 39, wherein the saccharification is conducted at a pHof 3 to 5.5 and at a temperature of 60-80° C. for 24 to 72 hours.
 44. Amethod of saccharifying a liquefied starch solution, which methodcomprises: (i) a saccharification wherein one or more enzymaticsaccharification stages takes place, and (ii) one or more hightemperature membrane separation steps, wherein the enzymaticsaccharification is carried out using a glucoamylase variant of claim
 2645. A method for altering the thermostability and/or for altering thespecific activity of a parent glucoamylase having at least 70% identityto the amino acid sequence published as SEQ ID NO: 2 in WO 00/04136 bymaking one or more mutation(s) in one or more of the followingposition(s) or region(s) of the parent glucoamylase corresponding to theposition(s) or region(s) of the amino acid sequence shown in SEQ ID NO:2 of WO 00/04136: a) said mutation(s) comprising one or moreinsertion(s) in: Region: 1-35, Region: 40-58, Region: 60-62, Region:73-80, Position: 93, Region: 95-101, Region: 103-121, Region: 123-124,Region: 126-127, Region: 170-175, Region: 177-184, Region: 200-212,Region: 234-246, Region: 287-312, Region: 314-319, Region: 334-339,Position: 341, Region: 354-356, Position: 358, Region: 360-374, Region:388-392, Position: 94, Region: 396-401, Region: 403407, Region: 409-414,Region: 445449, Region: 452467, Region: 469-470, or b) said mutation(s)comprising one or more substitution(s), insertion(s) and/or deletion(s)in: Region: 36-39, Region: 63-65, Position 67, Region: 69-71, Region:81-92, Region: 128-169, Region: 85-188, Region: 190-199, Region:213-222, Region: 224-226, Region: 228-233, Region: 247-271, Region:273-286, Region: 320-333, Region: 343-344, Region: 346-347, Region:349-351, Region: 375-378, Region: 380-385, Position: 387, Position: 415,Region: 417-424, Position: 426, Region: 428443, Region: 471485, Region:487-489, Region: 491-493, Region: 495-616, except the following aminoacid substitutions: A39V, P128S, G137A, G139A, D153N, S185H, G251A,D257E, E259D, E259Q, C320A, D375C, G383A, W417F, S431C, A435P, S436P,W437F, A442T, A471C, A479C, T480C, P481C, A495T and A495P.